Affinity labeling of the essential lysyl residue at the active site of enzymes by using nucleotide polyphosphate pyridoxal

We have discovered a new type of affinity labeling reagents for the nucleotide-binding site of protein by introducing an active site-directing moiety to pyridoxal 5-phosphate. Uridine diphosphopyridoxal almost completely inactivated glycogen synthase in a time-dependent manner (K _inact =25 µM;k ₀=0.22 min^–1). The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator Glc-6-P. The addition of cysteamine to the inactivated enzyme restored the original activity, whereas the treatment of the inactivated enzyme with NaBH₄ resulted in the fixation of the label to the enzyme protein. A peptide containing the label was isolated after proteolysis, and sequenced as E-V-A-K*-V-G-G-I-(Y). Adenosine polyphosphopyridoxal considerably inactivated lactate dehydrogenase in a time-dependent manner. The degree of inactivation was dependent on the number of phosphate groups; 64% (N=2), 51% (N=3), and 35% (N=4) at a reagent concentration of 1 mM for 30 min. The inactivation was protected by NADH, but not by pyruvate. Although the inactivation was not completed, the reagent was stoichiometrically incorporated into enzyme protein concomitantly with the inactivation. Affinity chromatographic analysis of the inactivated enzyme of Blue-Toyopearl revealed the presence of several protein species. The ratio of those species changed according to the stage of inactivation.